In total internal reflection microscope, the refractive behavior of light when transitioning from an optically denser to an optically less dense medium is utilized. For the transition from coverslip (n1=1.518) to water (n2=1.33), for example, the result is a critical angle of 61°, or the total reflection angle. Under total reflection conditions (angle=61°), a standing evanescent wave forms in the medium having the lower refractive index. The intensity of this wave decreases exponentially with distance from the interface. As a result of this, fluorophores located farther from the interface are not excited. The background fluorescence is considerably reduced, thereby improving image contrast and at the same time greatly raising resolution. A prerequisite for utilization of the phenomenon described above is a sufficiently large difference in refractive index between the coverslip and medium.
US 2002/0097489 A1 discloses a microscope with evanescent illumination of a specimen. The microscope contains a white-light source whose light is coupled, for evanescent illumination, via a slit stop through the microscope objective into the specimen slide carrying a specimen. The illuminating light propagates in the specimen slide by total internal reflection, illumination of the specimen occurring only in the region of the evanescent field projecting out of the specimen slide. Microscopes of this kind are known as TIRFM (total internal reflection fluorescent microscopes).
Because the evanescent field projects only approximately 100 nm into the specimen, the Z resolution of TIRF microscopes is extraordinarily good.
DE 101 08 796 A1 discloses a high-aperture objective, in particular for TIRF applications. The objective is made up of a first lens having positive refractive power and a second lens having negative refractive power, the focal length ratio between the two lenses being in the range from −0.4 to −0.1, and the total refractive power being greater than zero. The objective furthermore contains two positive lenses whose ratio of diameter to focal length is greater than 0.3 and less than 0.6. The objective furthermore contains a negative lens and a converging lens; the negative lens faces toward the front group and the focal length ratio of the negative lens and the converging lens is between −0.5 and −2.
DE 102 17 098 A1 discloses an incident illumination arrangement for TIRF microscopy. The incident illumination arrangement contains an illumination source that, in operation, emits a polarized illumination ray bundle that propagates at an angle to the optical axis, and a deflection device that deflects the illumination ray bundle and couples it into the objective parallel to the optical axis. In this incident illumination arrangement, provision is made for the illumination ray bundle emitted by the illumination source to have s and p polarization directions with a phase difference, and for the deflection device to reflect the illumination ray bundle x times, where x=((n×180°−d)/60°).
DE 101 43 481 A1 likewise discloses a microscope for total internal reflection microscopy (TIRM). The microscope comprises a microscope housing and an objective. The illuminating light proceeding from an illumination device can be coupled in via an adapter insertable into the microscope housing.
US 2004/0001253 A1 discloses a microscope having an optical illumination system that allows an easy switchover between evanescent illumination and reflected illumination. The illumination system contains a laser light source whose light is coupled into an optical fiber. Also provided is an outcoupling optical system that focuses the light emerging from the fiber into a rear focal point of the microscope objective. The optical fiber is displaceable in a plane perpendicular to the optical axis of the microscope objective.
DE 102 29 935 A1 discloses a device for coupling light into a microscope in which, in the field diaphragm plane, laser light is directed onto the sample through a light-guiding fiber incoupling system embodied as a slider. This is suitable in particular for the TIRF method.
In the investigation of biological specimens in particular, especially the investigation of the molecular structure of cells, there exists a need to ascertain the orientation of the molecules or dipoles in the specimen. It would be particularly advantageous if such orientations could be ascertained using apparatuses that are already in use in any case for investigation of the biological specimens.